Substituted Porphyrins

ABSTRACT

The present invention relates, in general, to a method of modulating physiological and pathological processes and, in particular, to a method of modulating cellular levels of oxidants and thereby processes in which such oxidants are a participant. The invention also relates to compounds and compositions suitable for use in such methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 11/127,302,filed May 12, 2005, which is a continuation of application Ser. No.09/880,125, filed Jun. 14, 2001, now U.S. Pat. No. 6,916,799, which is acontinuation of application Ser. No. 09/184,982, filed Nov. 3, 1988, nowabandoned, which claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application Ser. No. 60/064,116, filed Nov. 3, 1997,now expired. Each of the foregoing applications is incorporated byreference herein in its entirety.

TECHNICAL FIELD

The present invention relates, in general, to a method of modulatingphysiological and pathological processes and, in particular, to a methodof modulating cellular levels of oxidants and thereby processes in whichsuch oxidants are a participant. The invention also relates to compoundsand compositions suitable for use in such methods.

BACKGROUND

Oxidants are produced as part of the normal metabolism of all cells butalso are an important component of the pathogenesis of many diseaseprocesses. Reactive oxygen species, for example, are critical elementsof the pathogenesis of diseases of the lung, the central nervous systemand skeletal muscle. Oxygen free radicals also play a role in modulatingthe effects of nitric oxide (NO.). In this context, they contribute tothe pathogenesis of vascular disorders, inflammatory diseases and theaging process.

A critical balance of defensive enzymes against oxidants is required tomaintain normal cell and organ function. Superoxide dismutases (SODS)are a family of metalloenzymes that catalyze the intra- andextracellular conversion of O₂ ⁻ into H₂O₂ plus O₂, and represent thefirst line of defense against the detrimental effects of superoxideradicals. Mammals produce three distinct SODs. One is a dimeric copper-and zinc-containing enzyme (CuZn SOD) found in the cytosol of all cells.A second is a tetrameric manganese-containing SOD (Mn SOD) found withinmitochondria, and the third is a tetrameric, glycosylated, copper- andzinc-containing enzyme (EC-SOD) found in the extracellular fluids andbound to the extracellular matrix. Several other important antioxidantenzymes are known to exist within cells, including catalase andglutathione peroxidase. While extracellular fluids and the extracellularmatrix contain only small amounts of these enzymes, other extracellularantioxidants are also known to be present, including radical scavengersand inhibitors of lipid peroxidation, such as ascorbic acid, uric acid,and α-tocopherol (Halliwell et al, Arch. Biochem. Biophys. 280:1(1990)).

The present invention relates generally to low molecular weightporphyrin compounds suitable for use in modulating intra- andextracellular processes in which superoxide radicals, or other oxidantssuch as hydrogen peroxide or peroxynitrite, are a participant. Thecompounds and methods of the invention find application in variousphysiologic and pathologic processes in which oxidative stress plays arole.

SUMMARY OF THE INVENTION

The present invention relates to a method of modulating intra- orextracellular levels of oxidants such as superoxide radicals, hydrogenperoxide, peroxynitrite, lipid peroxides, hydroxyl radicals and thiylradicals. More particularly, the invention relates to a method ofmodulating normal or pathological processes involving superoxideradicals, hydrogen peroxide, nitric oxide or peroxynitrite, using lowmolecular weight antioxidants, and to methine (ie, meso) substitutedporphyrins suitable for use in such a method. The substituted porphyrinsare also expected to have activity as antibacterial and antiviralagents, and as ionophores and chemotherapeutics. Objects and advantagesof the present invention will be clear from the description thatfollows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Mechanism.

FIG. 2. Manganese meso-tetrakis-N-alkyl-pyridinium based porphyrins.

FIG. 3. SOD activity in vivo (E. coli) of 1, 2, 3* and 4* (20 μM) inminimal medium (mixture of atropoisomers, JI=SOD deficient strain, ABparental strain).

FIG. 4. Structures of MnCl_(x)TE-2-PyP⁵⁺ (x=1 to 4).

FIG. 5. ¹H-NMR spectrum (porphyrin ring) of H₂Cl_(2a)T-2-PyP in CDCl₃(δ=7.24 ppm). The four protons in alpha position of the four pyridylnitrogens are taken as integration reference.

FIG. 6. Plot of the free energy of activation (ΔG^(#)) for the O₂ ⁻dismutation reaction catalyzed by MnCl_(x)TE-2-PyP⁵⁺ as a function ofthe ground state free energy change (ΔG^(o)) for MnCl_(x)TE-2-PyP⁵⁺redox. ΔG^(#) and ΔG^(o) were calculated from k_(cat) and E^(o) _(1/2)values reported in Table 4 (F, R, h and k_(B) are Farday, molar gas,Planck and Boltzmann constants, respectively).

Numbers 0-4 correspond to x in MnCl_(x)TE-2-PyP⁵⁻

Corresponding data for one active site of Cu,Zn-SOD (Ellerby et al, J.Am. Chem. Soc. 1118:6556 (1996)).

FIG. 7. Illustrated are the chemical structures of three classes ofantioxidants. A) The meso-porphyrin class is depicted where: R₁ iseither a benzoic acid (tetrakis-(4-benzoic acid) porphyrin (TBAP)) or aN-methyl group in the 2 or 4 position of thepyridyl(tetrakis-(N-methylpyridinium-2(4)-yl) porphyrin (TM-2-PyP,TM-4-PyP)); R₂ is either a hydrogen (H) or a bromide (Br, OBTM-4-PyP)and where the porphyrin is ligated with either a manganese (Mn), cobalt(Co), iron (Fe), or zinc (Zn) metal. B) The vitamin E analog class isrepresented by trolox. C) The flavanoid class is represented by rutin.

FIG. 8. The time course of iron/ascorbate mediated oxidation of ratbrain homogenates. Rat brain homogenates were incubated for varioustimes with 0.25 μM FeCl₂ and 1 μM ascorbate, and lipid peroxidation wasmeasured as thiobarbituric acid reactive species (TBARS)spectrophotometrically at 535 nm (n=3).

FIG. 9. The comparison of trolox (▪), rutin (▴), bovine CuZnSOD (●),MnOBTM-4-PyP (▾) and MnTM-2-PyP (♦) in their ability to inhibitiron/ascorbate mediated oxidation of rat brain homogenates. Rat brainhomogenates were incubated for 30 minutes with 0.25 μM FeCl₂ and 1 μMascorbate, and lipid peroxidation was measured as thiobarbituric acidreactive species. The amount of TBARS formed in 30 minutes was expressedas 100% lipid peroxidation (n=3-6). Sigmoidal dose response curves werederived from fitting the data to a non-linear regression program.

FIG. 10. The comparison of manganic (▴), cobalt (●), iron (▾) and zinc(▪) analogs of TBAP in their ability to inhibit iron/ascorbate mediatedoxidation of rat brain homogenates. Rat brain homogenates were incubatedfor 30 minutes with 0.25 μM FeCl₂ and 1 μM ascorbate, and lipidperoxidation was measured as thiobarbituric acid reactive species. Theamount of TBARS formed in 30 minutes was expressed as 100% lipidperoxidation (n=36). Sigmoidal dose response curves were derived fromfitting the data to a non-linear regression program.

FIG. 11. The comparison of manganic (solid) and zinc (open) analogs ofTM-4-PyP (squares) and TM-2-PyP (triangles) in their ability to inhibitiron/ascorbate mediated oxidation of rat brain homogenates. Rat brainhomogenates were incubated for 30 minutes with 0.25 μM FeCl₂ and 1 μMascorbate, and lipid peroxidation was measured as thiobarbituric acidreactive species. The amount of TBARS formed in 30 minutes was expressedas 100% lipid peroxidation (n=3-6). Sigmoidal dose response curves werederived from fitting the data to a non-linear regression program.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of protecting against thedeleterious effects of oxidants, particularly, superoxide radicals,hydrogen peroxide and peroxynitrite, and to methods of preventing andtreating diseases and disorders that involve or result from oxidantstress. The invention also relates methods of modulating biologicalprocesses involving oxidants, including superoxide radicals, hydrogenperoxide, nitric oxide and peroxynitrite. The invention-further relatesto compounds and compositions, including low molecular weightantioxidants (eg mimetics of scavengers of reactive oxygen species,including mimetics of SODs, catalases and peroxidases) and formulationsthereof, suitable for use in such methods.

Mimetics of scavengers of reactive oxygen species appropriate for use inthe present methods include methine (ie meso) substituted porphines, orpharmaceutically acceptable salts thereof. The invention includes bothmetal-free and metal-bound porphines. In the case of metal-boundporphines, manganic derivatives of methine (meso) substituted porphinesare preferred, however, metals other than manganese, such as iron (II orIII), copper (I or II), cobalt (II or III), or nickel (I or II), canalso be used. It will be appreciated that the metal selected can havevarious valence states, for example, manganese II, III or V can be used.Zinc (II) can also be used even though it does not undergo a valencechange and therefore will not directly scavenge superoxide. The choiceof the metal can affect selectivity of the oxygen species that isscavenged. Iron-bound porphines, for example, can be used to scavengeNO. while manganese-bound porphines cannot. These metal bound porphinesscavenge peroxynitrite; iron, nickel and cobalt bound porphines tend tohave the highest reactivity with peroxynitrite.

Preferred mimetics of the invention are of Formula I or II:

or pharmaceutically acceptable salt thereof, wherein R is C₁-C₃ alkyl,preferably, C₁-C₄ alkyl, more preferably, methyl, ethyl or isopropyl,most preferably methyl. This mimetic can also be present metal-free orbound to a metal other than Mn. All atropoisomers of the above arewithin the scope of the invention, present in isolated form or as amixture of at least two. Atropoisomers wherein at least 3, preferably 4,of the R groups are above the porphyrin ring plane can be particularlyadvantageous.

One or more of the pyrrole rings of the porphyrin of Formula I or II canbe substituted at any or all beta carbons, ie: 2, 3, 7, 8, 12, 13, 17 or18. Such substituents, designated P, can be an electron withdrawinggroup, for example, each P can, independently, be a NO₂ group, a halogen(eg Cl, Br or F), a nitrile, a vinyl group, or a formyl group. Forexample, there can be 1, 2, 3, 4, 5, 6, 7 or 8 halogen (eg Br)substituents (when there are less than 8 halogen substituents, theremaining P's are advantageously hydrogen). Such substituents alter theredox potential of the porphyrin and thus enhance its ability toscavenge oxygen radicals. Each P can, independently, also be hydrogen.When P is formyl, it is preferred that there be not more than 2 (on nonadjacent carbons), more preferably 1, the remaining P's being hydrogen.When P is NO₂, it is preferred that there be not more than 4 (on nonadjacent carbons), more preferably 1 or 2, the remaining P's beinghydrogen.

Mimetics suitable for use in the present methods can be selected byassaying for SOD, catalase and/or peroxidase activity and stability.Mimetics can also be screened for their ability to inhibit lipidperoxidation in tissue homogenates using iron and ascorbate to initiatethe lipid peroxidation and measuring the formation of thiobarbituricacid reactive species (TBARS) (Ohkawa et al, Anal. Biochem. 95:351(1979) and Yue et al, J. Pharmacol. Exp. Ther. 263:92 (1992)). Theselective, reversible and SOD-sensitive inactivation of aconitase byknown O⁻ ₂ generators can be used as a marker of intracellular O⁻ ₂generation. Thus, suitable mimetics can be selected by assaying for theability to protect aconitase activity.

SOD activity can be monitored in the presence and absence of EDTA usingthe method of McCord and Fridovich (J. Biol. Chem. 244:6049 (1969)). Theefficacy of a mimetic can also be determined by measuring the effect ofthe mimetic on the aerobic growth of a SOD null E. coli strain versus aparental strain lacking the specific mutations. Specifically, parentalE. coli (AB1157) and SOD null E. coli. (JI132) can be grown in M9 mediumcontaining 0.2% casamino acids and 0.2% glucose at pH 7.0 and 37° C.;growth can be monitored in terms of turbidity followed at 700 nm. Thisassay can be made more selective for SOD mimetics by omitting thebranched chain, aromatic and sulphur containing amino acids from themedium (glucose minimal medium (M9), plus 5 essential amino acids) (seeExample V).

Efficacy of active mimetics can also be assessed by determining theirability to protect mammalian cells against methylviologen(paraquat)-induced toxicity. Specifically, rat L2 cells grown asdescribed below and seeded into 24 well dishes can be pre-incubated withvarious concentrations of the SOD mimetic and then incubated with aconcentration of methylviologen previously shown to produce an LC₇₅ incontrol L2 cells. Efficacy of the mimetic can be correlated with adecrease in the methylviologen-induced LDH release (St. Clair et al,FEBS Lett. 293:199 (1991)).

The efficacy of SOD mimetics can be tested in vivo with mouse and/or ratmodels using both aerosol administration and parenteral injection. Forexample, male Balb/c mice can be randomized into 4 groups of 8 mice eachto form a standard 2×2 contingency statistical model. Animals can betreated with either paraquat (40 mg/kg, ip) or saline and treated withSOD mimetic or vehicle control. Lung injury can be assessed 48 hoursafter paraquat treatment by analysis of bronchioalveolar lavage fluid(BALF) damage parameters (LDH, protein and % PMN) as previouslydescribed (Hampson et al, Tox. Appl. Pharm. 98:206 (1989); Day et al, J.Pharm. Methods 24:1 (1990)). Lungs from 2 mice of each group can beinstillation-fixed with 4% paraformaldehyde and processed forhistopathology at the light microscopic level.

Catalase activity can be monitored by measuring absorbance at 240 nm inthe presence of hydrogen peroxide (see Beers and Sizer, J. Biol. Chem.195:133 (1952)) or by measuring oxygen evolution with a Clark oxygenelectrode (Del Rio et al, Anal. Biochem. 80:409 (1977)). Peroxidaseactivity can be measured spectrophotometrically as previously describedby Putter and Becker: Peroxidases. In: Methods of Enzymatic Analysis, H.U. Bergmeyer (ed.), Verlag Chemie, Weinheim, pp. 286-292 (1983).Aconitase activity can be measured as described by Gardner and Fridovich(J. Biol. Chem. 260:19328 (1991)). The ability of mimetics to inhibitlipid peroxidation is assessed as described by Ohkawa et al (Anal.Biochem. 95:351 (1979)) and Yue et al (J. Pharmacol. Exp. Ther. 263:92(1992)).

Active mimetics can be tested for toxicity in mammalian cell culture bymeasuring lactate dehydrogenase (LDH) release. Specifically, rat L2cells (a lung Type II like cell; (Kaighn and Douglas, J. Cell Biol.59:160a (1973)) can be grown in Ham's F-12 medium with 10% fetal calfserum supplement at pH 7.4 and 37° C.; cells can be seeded at equaldensities in 24 well culture dishes and grown to approximately 90%confluence; SOD mimetics can be added to the cells at log doses (egmicromolar doses in minimal essential medium (MEM)) and incubated for 24hours. Toxicity can be assessed by morphology and by measuring therelease of the cytosolic injury marker, LDH (eg on a thermokinetic platereader), as described by Vassault (In: Methods of Enzymatic Analysis,Bergmeyer (ed) pp. 118-26 (1983); oxidation of NADH is measured at 340nm).

Synthesis of mimetics suitable for use in the present method can beeffected using art-recognized protocols (see also Examples I, II, IIIand Iv and Sastry et al, Anal. Chem. 41:857 (1969), Pasternack et al,Biochem. 22:2406 (1983); Richards et al, Inorg. Chem. 35:1940 (1996) andU.S. application Ser. No. 08/663,028, particularly the details thereinrelating to syntheses). Separation of atropoisomers can be effectedusing a variety of techniques.

One specific embodiment of the present invention relates to a method ofregulating NO. levels by targeting the above-described porphines tostrategic locations. NO. is an intercellular signal and, as such, NO.must traverse the extracellular matrix to exert its effects. NO.,however, is highly sensitive to inactivation mediated by O₂ ⁻ present inthe extracellular spaces. The methine (meso) substituted porphyrins ofthe invention can increase bioavailability of NO. by preventing itsdegradation by O₂ ⁻.

In a further embodiment, the mimetics of the invention are used ascatalytic scavengers of reactive oxygen species to protect againstischemia reperfusion injuries associated with myocardial infarction,stroke, acute head trauma, organ reperfusion following transplantation,bowel ischemia, hemorrhagic shock, pulmonary infarction, surgicalocclusion of blood flow, and soft tissue injury. The mimetics canfurther be used to protect against skeletal muscle reperfusion injuries.The mimetics can also be used to protect against damage to the eye dueto sunlight (and to the skin) as well as glaucoma, and maculardegeneration in the eye. The mimetics can also be used to protectagainst and/or treat cataracts. The mimetics can also be used to protectagainst and/or treat inflammatory diseases of the skin (e.g.,psoriasis). Diseases of the bone are also amenable to treatment with themimetics. Further, connective tissue disorders associated with defectsin collagen synthesis or degradation can be expected to be susceptibleto treatment with the present mimetics, as should the generalizeddeficits of aging.

In yet another embodiment, the mimetics of the invention can be used ascatalytic scavengers of reactive oxygen species to increase the verylimited storage viability of transplanted hearts, kidneys, skin andother organs and tissues. The invention also provides methods ofinhibiting damage due to autoxidation of substances resulting in theformation of O₂ ⁻ including food products, pharmaceuticals, storedblood, etc. To effect this end, the mimetics of the invention are addedto food products, pharmaceuticals, stored blood and the like, in anamount sufficient to inhibit or prevent oxidation damage and thereby toinhibit or prevent the degradation associated with the autoxidationreactions. (For other uses of the mimetics of the invention, see U.S.Pat. No. 5,227,405). The amount of mimetic to be used in a particulartreatment or to be associated with a particular substance can bedetermined by one skilled in the art.

In yet another embodiment, the mimetics of the invention can be used toscavenge hydrogen peroxide and thus protect against formation of thehighly reactive hydroxyl radical by interfering with Fenton chemistry(Aruoma and Halliwell, Biochem. J. 241:273 (1987); Mello Filho et al,Biochem. J. 218:273 (1984); Rush and Bielski, J. Phys. Chem. 89:5062(1985)). The mimetics of the invention may also be used to scavengeperoxynitrite, as demonstrated indirectly by inhibition of the oxidationof dihydrorhodamine 123 to rhodamine 123 and directly by acceleratingperoxynitrite degradation by stop flow analysis.

Further examples of specific diseases/disorders appropriate fortreatment using the mimetics of the present invention include diseasesof the central nervous system (including AIDS dementia, stroke,amyotrophic lateral sclerosis (ALS), Parkinson's disease andHuntington's disease) and diseases of the musculature (includingdiaphramic diseases (eg respiratory fatigue in emphysema, bronchitis andcystic fibrosis), cardiac fatigue of congestive heart failure, muscleweakness syndromes associated with myopathies, ALS and multiplesclerosis). Many neurologic disorders (including stroke, Huntington'sdisease, Parkinson's disease, ALS, Alzheimer's and AIDS dementia) areassociated with an over stimulation of the major subtype of glutamatereceptor, the NMDA (or N-methyl-D-aspartate) subtype. On stimulation ofthe NMDA receptor, excessive neuronal calcium concentrations contributeto a series of membrane and cytoplasmic events leading to production ofoxygen free radicals and nitric oxide (NO.). Interactions between oxygenfree radicals and NO. have been shown to contribute to neuronal celldeath. Well-established neuronal cortical culture models ofNMDA-toxicity have been developed and used as the basis for drugdevelopment. In these same systems, the mimetics of the presentinvention inhibit NMDA-induced injury. The formation of O⁻ ₂ radicals isan obligate step in the intracellular events culminating in excitotoxicdeath of cortical neurons and further demonstrate that the mimetics ofthe invention can be used to scavenge O⁻ ₂ radicals and thereby serve asprotectants against excitotoxic injury.

The present invention also relates to methods of treating AIDS. TheNfKappa B promoter is used by the HIV virus for replication. Thispromoter is redox sensitive, therefore, an antioxidant can regulate thisprocess. This has been previously shown for two metalloporphyrinsdistinct from those of the present invention (Song et al, AntiviralChem. And Chemother. 8:85 (1997)). The invention also relates to methodsof treating arthritis, systemic hypertension, atherosclerosis, edema,septic shock, pulmonary hypertension, including primary pulmonaryhypertension, impotence, MED, infertility, endometriosis, prematureuterine contractions, microbial infections, gout and in the treatment ofType I and Type II diabetes mellitus. The mimetics of the invention canbe used to ameliorate the toxic effects associated with endotoxin, forexample, by preserving vascular tone and preventing multi-organ systemdamage.

Inflammations, particularly inflammations of the lung, are amenable totreatment using the present invention (note particularly theinflammatory based disorders of asthma, ARDS including oxygen toxicity,pneumonia (especially AIDS-related pneumonia), cystic fibrosis, chronicsinusitis and autoimmune diseases (such as rheumatoid arthritis)).EC-SOD is localized in the interstitial spaces surrounding airways andvasculature smooth muscle cells. EC-SOD and O₂ ⁻ mediate theantiinflammatory—proinflammatory balance in the alveolar septum. NO.released by alveolar septal cells acts to suppress inflammation unlessit reacts with O₂ ⁻ to form ONOO⁻. By scavenging O₂ ⁻, EC-SOD tips thebalance in the alveolar septum against inflammation. Significant amountsof ONOO⁻ will form only when EC-SOD is deficient or when there isgreatly increased O₂ ⁻ release. Mimetics described herein can be used toprotect against destruction caused by hyperoxia.

The invention further relates to methods of treating memory disorders.It is believed that nitric oxide is a neurotransmitter involved inlong-term memory potentiation. Using an EC-SOD knocked-out mouse model(Carlsson et al, Proc. Natl. Acad. Sci. USA 92:6264 (1995)), it can beshown that learning impairment correlates with reduced superoxidescavenging in extracellular spaces of the brain. Reduced scavengingresults in higher extracellular 0-2 levels. O⁻ ₂ is believed to reactwith nitric oxide thereby preventing or inhibiting nitricoxide-medicated neurotransmission and thus long-term memorypotentiation. The mimetics of the invention can be used to treatdementias and memory/learning disorders.

The availability of the mimetics of the invention also makes possiblestudies of processes mediated by O₂ ⁻, hydrogen peroxide, nitric oxideand peroxynitrite.

The mimetics described above can be formulated into pharmaceuticalcompositions suitable for use in the present methods. Such compositionsinclude the active agent (mimetic) together with a pharmaceuticallyacceptable carrier, excipient or diluent. The composition can be presentin dosage unit form for example, tablets, capsules or suppositories. Thecomposition can also be in the form of a sterile solution suitable forinjection or nebulization. Compositions can also be in a form suitablefor opthalmic use. The invention also includes compositions formulatedfor topical administration, such compositions taking the form, forexample, of a lotion, cream, gel or ointment. The concentration ofactive agent to be included in the composition can be selected based onthe nature of the agent, the dosage regimen and the result sought.

The dosage of the composition of the invention to be administered can bedetermined without undue experimentation and will be dependent uponvarious factors including the nature of the active agent, the route ofadministration, the patient, and the result sought to be achieved. Asuitable dosage of mimetic to be administered, for example, IV ortopically, can be expected to be in the range of about 0.01 to 100mg/kg/day, preferably 0.1 to 10 mg/kg/day. For aerosol administration,it is expected that doses will be in the range of 0.01 to 1.0 mg/kg/day.Suitable doses of mimetics will vary, for example, with the mimetic andwith the result sought. The results of Faulkner et al (J. Biol. Chem.269:23471 (1994)) indicate that the in vivo oxidoreductase activity ofthe mimetics is such that a pharmaceutically effective dose will be lowenough to avoid problems of toxicity. Doses that can be used includethose in the range of 1 to 50 mg/kg.

Certain aspects of the present invention will be described in greaterdetail in the non-limiting Examples that follow.

EXAMPLES

The following chemicals were utilized in Examples I-V that follow.

The chloride salts of ortho and meta metal-free ligands (H₂TM-2-PyPCl₅and H₂TM-3-PyPCl₅) were purchased from MidCentury Chemicals, and thetosylate salts of the para metal-free ligand H₂TM-4-PyP (CH₅PhSO₃)₅)were purchased from Porphyrin Products. The purity was checked in termsof elemental analysis and spectral properties, ie, molar absorptivitiesand corresponding wave-length of the Soret bands. The Soret bandproperties of metal-free ligands were ε_(413.3nm)=2.16×10⁵M⁻¹ cm⁻¹(H₂TM-2-PyPCl₄), ε_(416.6nm)=3.18×10⁵ M⁻¹cm⁻¹ (H₂TM-3-PyPCl₄),ε_(422.0nm)=2.35×10⁵ M⁻¹ cm⁻¹ (H₂TM-4-PyPCl₄). The non-methylated orthometal-free ligand (H₂T-2-PYP) was bought from MidCentury Chemicals andthe purity checked in terms of elemental analysis (see below).Iodoethane, 1-iodobutane, anhydrous manganese chloride (MnCl₂),MnCl₂.4H₂O, tetrabutylammonium chloride (TBA) and ammoniumhexaafluorophosphate (PF₆NH₄) were purchased from Aldrich.

Example I Synthesis of meso-tetrakis-(N-methylpyridinium-2-yl)porphyrinand meso-tetrakis-(N-methylpyridinium-3-yl)porphyrin

Metal-free porphyrins meso-tetrakis-(2-pyridyl)porphyrin (H₂T-2-PyP) andmeso-tetrakis-(3-pyridyl)porphyrin (H₂T-3-PyP) were synthesized viaRothmund condensation with use of a modified Adler procedure(Kalyanasundaram, Inorg. Chem. 23:2453 (1984); (Torrens et al, J. Am.Chem. Soc. 94:4160 (1972)). Into a 100 mL refluxing solution ofpropionic acid were slowly injected equimolar amounts of freshlydistilled pyrrole and pyridine-2- or pyridine-3-carboxyaldehyde, and thesolution was allowed to reflux for about 45 min, after which thepropionic acid was distilled off. The black residues were neutralizedwith NaOH, washed with methanol, dissolved in CH₂Cl₂ (dichloromethane)and chromotographed on a neutral Woelm alumina column prepared withacetone. After elution of a pale blue fraction, H₂TPyP was eluted withthe use of CH₂Cl₂ containing 5-10% of pyridine. Shiny dark purplecrystals were recovered from the dark red eluant after removal ofsolvents on rotavaporator. Methylation of H₂TPyPs was carried using theexcess of methyl-p-toluenesulfonate in refluxing chloroform(Kalyanasundaram, Inorg. Chem. 23:2453 (1984); (Hambright et al, Inorg.Chem. 15:2314 (1976)). Both of the alkylated porphyrins spontaneouslyprecipitated from hot chloroform solutions and were washed with etherand air dried.

Example II Preparation of Manganese Complexes of Ortho, Meta and ParaIsomers of H₂TMPyP⁴⁺

The metallation was performed in water at room temperature. Theporphyrin to metal ratio was 1:5 in the case of meta and ortho isomersand 1:14 in the case of para isomer. The solid MnCl₂×4H₂O (Aldrich) wasadded to the aqueous metal-free porphyrins after the pH of the solutionwas brought to ˜pH=10.2. The metallation was completed inside an hour inthe cases of all three isomers. For the preparation of ortho and metacompounds, MnTM-2-PyP⁵⁺ and MnTM-3-PyP⁵⁺, 300 mg of the metal-freeligand, either H₂TM-2-PyP⁴⁺ or H₂TM-3-PyP⁴⁺, was dissolved in 100 mLwater, pH brought to 10.2 with several drops of 1M NaOH, followed by theaddition of 340 mg of MnCl₂. The metallation was followed spectrallythrough the disappearance of the Soret band of H₂TM-2-PyP⁴⁺ or H₂TM-3PyP⁴⁺ at 413.3 nm or 416.6 nm, respectively, and the appearance of theSoret bands of manganese complexes at 454.1 nm and 459.8 nm,respectively.

The excess of metal was eliminated as follows for all three (ortho, metaand para) isomers of MnTMPyP⁵⁺. The MnTMPyP⁵⁺ was precipitated as PF₆ ⁻salt by adding 50-fold excess of NH₄PF₆. The precipitate was washed with2-propanol:diethylether=1:1, and dried in vacuum at room temperature.Dry PF₆ ⁻ salt of MnTMPyP⁵⁻ was then dissolved in acetone (370 mg in 100mL acetone) and 1 g of tetrabutylammonium chloride added. Theprecipitate was washed with acetone and dried overnight in vacuum atroom temperature. In order to obtain a pure compound, the procedure wasrepeated. The elemental analysis was done for all metallated isomers.The compounds were analyzed in spectral terms and the following datawere obtained: Soret bands properties of metallated compounds were:ε_(454.1nm)=12.3×10⁴ cm⁻¹ M⁻¹ (MnTM-2-PyPCl₅), ε_(459.8nm)=13.3×10⁴ cm⁻¹M⁻¹ (MnTM-3-PyPCl₅), ε_(462.2nm)=13.9×10⁴ cm⁻¹ M⁻¹ (MnTM-4-PyPCl₅).

Metallation was performed in methanol as well. In addition, whenperformed in water, the metal:ligand ratio varied from 1:5, to 1:14 to1:100. Under all conditions, the given molar absorptivities wereobtained. The calculations were based on the metal-free ligands thatwere analyzed prior to metallation. The molar absorptivities of themetal-free ligands were consistent with literature as well as theirelemental analyses.

The elemental analyses of MnTM2-PyPCl₅ and MnTM-3-PyDCl₅ are shown inTable 1 TABLE 1 C* H* N* MnTM2-PyPCl₅•6 H₂O 52.99(52.90) 4.85(4.64)11.22(11.21) MnTM-3-PyPCl₅•3 H₂O 55.41(54.87) 4.97(4.40) 11.10(11.69)*Found (calcd).

Example III Synthesis of manganicmeso-tetrakis-(N-ethylpyridinium-2-yl)porphyrin

50 mg of H₂T-2-PyP was dissolved in 30 mL of anhydrous dimethylformamide(DMF) and the solution was stirred and heated at 100° C., 20 mg ofanhydrous MnCl₂ (20 eq) were added and the solution stirred for 3 days.The completion of the metallation was checked by UV spectroscopy. Uponmetallation, the temperature was decreased to 60° C., 0.65 mL ofiodoethane (100 eq) was added, and the solution was stirred for 7 days(Perree-Fauvet et al, Tetrahedron 52:13569 (1996)). DMF was evaporated,10 mL of acetone was added, and the product was precipitated adding 20mL of a solution of TEA in acetone (0.45 M); indeed, contrary to theiodide salt, the chloride salt precipitates in acetone. The product waspurified using the “double precipitation” method, as described above.The product was dried overnight in vacuum, over P₂O₅, at 70° C., leadingto 125 mg (95%) of a dark purple solid. UV (H₂O), ε_(454.0nm)=1.41×10⁵M⁻¹cm⁻¹. Elemental analysis, calcd. for MnC₄₈N₈H₄₄Cl₅.5H₂O: C (54.64), H(5.16), N (10.62); found: C (54.55), H (5.36), N (10.88).

Example IV Synthesis of manganicmeso-tetrakis-(N-butylpyridinium-2-yl)porphyrin

The same procedure described above was used. 0.92 mL of 1-iodobutane(100 eq) was added and the mixture stirred at 100° C. for 7 days. Dryingof the chloride salt resulted in 70 mg (50%) of a dark purple viscousproduct. The elemental analysis was thus performed on thehexafluorophosphate salt (non-viscous). The chlorine salt iswater-soluble (micelles were not observed). UV(H₂O) of the chloridesalt, ε_(454.0) 1.21×10⁵ M⁻¹cm⁻¹. Elemental analysis, calcd. ForMnC₅₆H₆₀N₈P₅F₃₀.H₂O: C (40.94), H (3.80), N (6.82); found: C (41.15), H(4.35), N (6.52).

Example V The ortho effect makes manganicmeso-tetrakis-(N-alkylpyridinium-2-yl)-porphyrin a powerful superoxidedismutase mimic

The superoxide dismutase activity of the mimetics of the inventiondepends on a number of factors, including thermodynamic factors (eg themetal-centered redox-potential see FIG. 1)), and kinetic factors (egelectrostatic facilitation). In an in vitro enzymatic assay of SODactivity (see McCord and Fridovich, J. Biol. Chem. 244:6049 (1969)), theortho compound “3” proves to be more than an order of magnitude moreactive than the para compound “1” (see FIG. 2 (note also Table 2 where“2” is the meta compound and “4” and “5” are ortho compounds that carry4 ethyl or 4 butyl groups, respectively)).

The activity in vivo of the mimetics of the invention can be tested onan E. coli strain deleted of the genes coding for both the MnSOD andFeSOD. In this assay, the efficacy of a mimetic is determined bymeasuring the effect of the mimetic on the aerobic growth of a SOD nullE. coli strain versus a parental strain. Specifically, parental E. coli(AB1157) and SOD null E. coli. (JI132) are grown in M9 medium containing0.2% casamino acids and 0.2% glucose at pH 7.0 and 37° C.; growth ismonitored in terms of turbidity followed at 700 nm. This assay is mademore selective for SOD mimetics by omitting the branched chain, aromaticand sulphur containing amino acids from the medium (glucose minimalmedium (M9), plus 5 essential amino acids). As shown in FIG. 3, theincrease in activity by the “ortho effect” was confirmed in that, underthese growth conditions, SOD null cells cultured in the presence ofcompound “1” did not show an increase in A₇₀₀ while such cells culturedin the presence of compounds “3” and “4” (as well as “2”) did.

The “ortho effect” also decreases the toxicity. It is well known thatporphyrins, and particularly cationic porphyrins, interact with DNA andcan act as DNA cleavers. This fact can be an issue in the use ofmetallo-porphyrins as anti-tumor drugs. The present mimetics avoid thisinteraction. In addition to the increase in activity, the interactionwith DNA of the meta “2” and the ortho “3” compounds, is greatlydecreased. This is clearly demonstrated by the measurements of the SODactivity in vitro in the presence of DNA (see Table 2), and by thedecreased toxicity in vivo (E. coli) (see FIG. 3).

In order to maximize the decrease in toxicity due to interaction withDNA, two derivatives of the ortho compound have been prepared whichcarry four ethyl or four butyl groups (“4” and “5”, respectively). Theethyl derivative “4” was significantly less toxic than the methylderivative “3” (see Table 2 and FIG. 3). However, in comparison to theethylated derivative “4”, the butylated derivative did not show afurther decrease in toxicity (see Table 2). These data indicate thatortho ethyl groups are sufficient to inhibit binding of the porphyrin toDNA. TABLE 2 δ_(SB) (nm) ε(10³) E_(1/2) (V) k_(cat) (M⁻¹s⁻¹) DNA-IC₅₀ 1 462.2 139 +0.060 3.3 10⁶ 7.0 10⁻⁶ 2  459.8 133 +0.042 4.1 10⁶ 2.2 10⁻⁵3* 454.0 123 4.5 10⁷ 3.3 10⁻⁵ 4* 454.0 141 4.5 10⁷ 6.7 10⁻⁵ 5* 454.0 1103.0 I0⁷ 6.7 10⁻¹Table. UV parameters, redox potential (vs NHE), SOD like activity andDNA interaction parameters of 1, 2, 3 and its atropisomers, 4 and 5(*mixture of atropisomers, δ_(SB) Soret band wave-lenght, ε = molecular# absortivity of the Soret band, E_(1/2) = one-electron metal-centeredredox-potential, k_(cat) = rate constant for the superoxide dismutatianreaction, DNA-IC₅₀ = concentration of DNA for 50% inhibition of thesuperoxide dismutatian reaction).

Example VI Syntheses and Superoxide Dismutating Activities of Partially(1 to 4) O-Chlorinated Derivatives of Manganese (III)Meso-tetrakis-(N-ethylpyridinium-2-yl)-Porphyrin

Materials and Methods

Materials. 5,19,15,20-Tetrakis-(2-pyridyl)-porphyrin (H₂T-2-PyP) waspurchased from Mid-Century chemicals (Posen, Ill.) (Torrens et al, J.Am. Chem. Soc. 94:4160 (1972)). N-Chlorosuccinimide (NCS),ethyl-p-toluenesulfonate (ETS), tetrabutylammonium chloride (98%)(TBAC), ammonium hexafluorophosphate (NH₄PF₆), manganese chloride,sodium L-ascorbate (99%), cytochrome c, xanthine,ethylenedinitrilotetraacetic acid (EDTA), N,N-dimethylformamide (98.8%,anhydrous) and 2-propanol (99.5%) were from Sigma-Aldrich. Ethanol(absolute), acetone, ethyl ether (anhydrous), chloroform anddichloromethane (HPLC grade) were from Mallinckrodt, and used withoutfurther purification. Xanthine oxidase was supplied by R. D. Wiley (Waudet al, Arch. Biochem. Biophys. 19:695 (1975)). Thin-layer chromatography(TLC) plates (Baker-flex silica gel IB) were from J. T. Baker(Phillipsburg, N.J.). Wakogel C-300 was from Wako Pure IndustryChemicals, Inc (Richmond, Va.).

Instrumentation. Proton nuclear magnetic resonance (¹H-NMR) spectra wererecorded on a Varian Inova 400 spectrometer. Ultravisible/visible(UV/VIS) spectra were recorded on a Shimadzu spectrophotometer ModelUV-260. Matrix-assisted laser desorption/ionization-time offlight—(MALDI-TOFMS) and electrospray/ionization (ESMS) massspectrometry were performed on a Bruker Proflex III™ and a FisonsInstruments VG Bio-Q triple quadrupole spectrometers, respectively.

H₂Cl₁T-2-PyP. 50 mg (8.1×10⁻⁵ moles) of H₂T-2-PyP was reflexed inchloroform with 43 mg (3.22×10⁻⁴ moles) of NCS (Ochsenbein et al, Angew.Chem. Int. Ed. Engl. 33:348 (1994). The reaction was followed by normalphase silica TLC using a mixture EtOH/CH₂Cl₂ (5:95) as eluant. After 6hours of reaction the solution was washed once with distilled water. Thechloroform was evaporated and the products of the reaction werechromatographed over 100 g of Wakogel C-300 on a 2.5×50 cm column usingthe same eluant. The fraction corresponding to H₂Cl₁T-2-PyP was purifiedagain using the same system leading to 16 mg of a black purple solid(30%). TLC: R_(f)=0.47. UV/VIS (CHCl₃): λ_(nm) (log ε) 419.6 (5.44),515.2 (4.21), 590.0 (3.72), 645.8 (3.25). MALDI-TOFMS: m/z=654 (M+H⁺).¹H-NMR (CDCl₃): δ_(ppm)−2.91 (2H, NH); 7.66-7.74 (m, 4H); 7.99-8.21 (m,8H); 7.68 (s, 1H); 8.74 (d, 1H, J=6 Hz); 8.76 (d, 1H, J=6 Hz); 8.76 (d,1H, J=6 Hz); 8.88 (d, 1H, J=6 Hz); 8.90 (d, 1H, J=6 Hz); 8.94 (d, 1H,J=6 Hz); 9.04-9.14 (m, 4H).

H₂Cl_(2a)T-2-PyP. The same procedure as described above, leading to 5.3mg of a black purple solid (10%). TLC: R_(f)=0.50. UV/VIS (CHCl₃):λ_(nm) (log ε) 421.4 (5.38), 517.8 (4.21), 591.4 (3.78), 647.6 (3.51).MALDI-TOFMS: m/z=688 (M+H⁺). ¹H-NMR (CDCl₃): δ_(ppm)−2.98 (2H, NH);7.66-7.74 (m, 4H); 8.00-8.20 (m, 8H); 8.70 (s, 2H); 8.82 (d, 2H, J=6Hz); 8.91 (d, 2H, J=6 Hz); 9.06-9.14 (m, 4H).

H₂Cl_(2b+2c)T-2-PyP. The same procedure leading to 11 mg of a blackpurple solid (20%). TLC: R_(f) 0.53. UV/VIS (CHCl₃): λ_(nm) (log ε)421.4 (5.42), 516.8 (4.25), 593.2 (3.74), 646.2 (3.31); MALDI-TOFMS,m/z=688 (M+H⁺). ¹H-NMR (CDCl₃): δ_(ppm)−3.04 (2H, NH); −2.84 (1H, NH);−2.87 (1H, NO; 7.66-7.74 (m, 8H); 7.98-8.20 (m, 16H); 8.59 (s, 1H); 8.61(s, 1H); 8.73 (d, 2H; J<2 Hz); 8:78 (d, 2H, J=6 Hz); 8.87 (d, 2H, J=6Hz); 8.93 (d, 2H, J<2 Hz); 9.02-9.14 (m, 8H).

H₂Cl₃T-2-PyP. The same procedure using 65 mg (4.87×10⁻⁴ moles) of NCS,leading to 8.4 mg of a black purple solid (14%). TLC: R_(f)=0.55 UV/VIS(CHCl₃): λ_(nm) (log ε) 422.8 (5.37), 519.4 (4.21), 593.8 (3.71), 651.4(3.37). MALDI-TOFMS: m/z=723 (M+H⁺). ¹H-NMR (CDCl₃): δ_(ppm)−3.08 (1H,NH); −3.15 (1H, NH); 7.66-7.74 (m, 4H); 8.00-8.18 (m, 8H); 8.56 (s, 1H),8.72 (d, 1H, J=6 Hz); 8.76 (d, 1H, J=6 Hz); 8.82 (d, 1H, J=6 Hz); 8.88(d, 1H, J=6 Hz); 9.04-9.14 (m, 4H).

H₂Cl₄T-2-PyP. The same procedure using 65 mg (4.87×10⁻⁴ moles) of NCS,leading to 7.3 mg of a black purple solid (12%). TLC: R_(f)=0.58. UV/VIS(CHCl₃): , (log ε) 423.4 (5.33), 520.0 (4.19), 595.6 (3.66), 651.0(3.33). MALDI-TOFMS: m/z=758 (M+H^(+).) ¹H-NMR (CDCl₃): δ_(ppm)−3.14(2H, NH); 7.66-7.74 (m, 4H); 7.98-8.16 (m, 8H); 8.74 (d, 4H, J<2 Hz);9.06-9.12 (m, 4H).

MnTE-2-PyP⁵⁺. 100 mg (1.62×10⁻⁴ moles) of H₂T-2-PyP was dissolved in 5mL of warm DMF (anhydrous), 5.5 mL (3.22×10⁻² moles) ofethyl-p-toluenesulfonate (ETS) was added under stirring at 90° C. andallowed to react for 2448 hours. The completion of tetra-N-ethylationwas followed by normal phase silica TLC using a mixtureKNO_(3sat)/H₂O/CH₃CN (1:1:8) as eluant (Batinic-Haberle et al, J. Biol.Chem. 273:24521 (1998)). Upon the completion of the reaction, the DMFwas removed in vacuo and 5 mL of acetone was then added. To thissolution, a concentrated solution of tetrabutylammonium chloride (TBAC)in acetone (˜1 g/10 mL acetone) was added dropwise under stirring untilprecipitation of the chloride was complete. The resulting purple solidwas dissolved in 10 mL of water, the pH of the solution was raised to 12with NaOH and 640 mg of MnCl₂.4H₂O (3.23×10⁻³ moles) was added(Batinic-Haberle et al, J. Biol. Chem. 273:24521 (1998). Upon completionof metallation, the pH was lowered between 4 and 7 in order tofacilitate the auto-oxidation of Mn(II) into Mn(III), and the excess ofmetal was eliminated as follows. The solution was filtered, and aconcentrated aqueous solution of NH₄ PF₆ was added to precipitate themetalloporphyrin as the PF₆— salt (Batinic-Haberle et al, Arch. Biochem.Biophy. 343:225 (1997); Richards et al, Inorg. Chem. 35:1940 (1996)).The precipitate was thoroughly washed with a mixture 2-propanol/ethylether (1:1), dried in vacuo at room temperature. The resulting solid wasthen dissolved in acetone and a concentrated solution of TBAC was addedto isolate the metalloporphyrin in the form of its chloride salt. Theprecipitate was washed thoroughly with acetone and dried in vacuo atroom temperature leading to 150 mg of a black red solid (95%). TLC:R_(f)=0.18. UV/VIS (H₂O): λ_(nm) (log δ) 364.0 (4.64), 453.8 (5.14),558.6 (4.05). ESMS: m/z=157.4 (M⁵⁺/5). Anal. calcd. forMnC₄₈N₈H₄₄Cl₅.5H₂O: C, 54.64; H, 5.16; N, 10.62. Found: C, 54.55; H,5.40; N, 10.39. (See FIG. 4 for compound structures).

MnCl₁TE-2-PyP⁵⁺. The same procedure as described above starting from 10mg (1.53×10−5 moles) of H₂Cl₁T-2-PyP and 0.5 mL (2.94×10⁻³ moles) of ETSin 1 mL of DMF. TLC: R_(f)=0.20. UV/VIS (H₂O): λ_(nm) (log ε) 365.6(4.63), 455.6 (5.13), 560.6 (4.02). ESMS: m/z=164.3 (M⁵⁺/5). Anal.calcd. for MnC₄₈N₈H₄₃Cl₆.5H₂O: C, 52.91; H, 4.90; N, 10.28. Found: C,52.59; H, 5.28; N, 10.14.

MnCl_(2a)TE-2-PyP⁵⁺. The same procedure starting from 5 mg (7.28×10⁻⁶moles) of H₂Cl_(2a)T-2-PyP and 0.25 mL (1.47×10⁻³ moles) of ETS, leadingto 7.5 mg of a black red solid (95%). TLC: R_(f)=0.21. UV/VIS (H₂O):λ_(nm) (log ε) 365.8 (4.58), 456.4 (5.05), 562.2 (4.00). ESMS: m/z=171.1(M⁵⁺/5). Anal. calcd. for MnC₄₈N₈H₄₂Cl₇.6H₂O: C, 50.48; H, 4.77; N,9.81. Found: C, 50.08; H, 4.60; N, 10.01.

MnCl_(2b+2c)TE-2-PyP⁵⁺. The same procedure starting from 5 mg (7.28×10⁻⁶moles) of H₂Cl_(2b+2c)T-2-PyP, leading to 7.5 mg of a black red solid(95%). TLC: R_(f)=0.22. UV/VIS (H₂O): λ_(nm) (log ε) 365.2 (4.63), 457.4(5.08), 462.2 (4.06). ESMS: m/z==171.1 (M⁵⁺/5). Anal. calcd. forMnC₄₈N₈H₄₂C₇.5H₂O: C, 51.29; H, 4.66; N, 9.97. Found: C, 51.31; H, 5.19;N, 9.68.

MnCl₃TE-2-PyP⁵⁺. The same procedure starting from 5 mg (6.93×10⁻⁶ moles)of H₂Cl₃T-2-PyP, leading to 7.5 mg of a black brown solid (95%). TLC:R_(f)=0.23. UV/VIS (H₂O): λ_(nm) (log ε) 364.8 (4.58), 458.0 (4.98),466.4 (4.00). ESMS: m/z=178.1 (M⁵⁺/5). Anal. calcd. forMnC₄₈N₃H₄₁C₈.6H₂O: C, 49.00; H, 4.54; N, 9.52. Found: C, 48.40; H, 4.26;N, 9.59.

MnCl₄TE-2-PyP⁵⁺. The same procedure starting from 5 mg (6.61×10⁻⁶ moles)of H₂Cl₄T-2-PyP, leading to 7.5 mg of a black brown solid (95%). TLC:R_(f)=0.24. UV/VIS (H₂O): λ_(nm) (log ε) 365.8 (4.52), 459.2 (4.90),567.0 (3.96). ESMS: m/z=184.9 (M⁵⁺/5). Anal. calcd. forMnC₄₈N₈H₄₀Cl₉.5H₂O: C, 48.33; H, 4.22; N, 9.39. Found: C, 48.38; H,4.45; N, 9.53.

Electrochemistry. The electrochemical characterization was performed asdescribed previously on a Voltammetric Analyzer Model 600 (CHinstrument) using a glassy carbon electrode (Ag/AgCl reference and Ptauxiliary electrodes), at 0.5 mM porphyrin, pH 7.8 (0.05 M phosphatebuffer), 0.1 M NaCl. The potentials were standardized against potassiumferricyanide/potassium ferrocyanide couple (Batinic-Haberle et al, Arch.Biochem. Biophys. 343:225 (1997); Kolthof et al, J. Phys. Chem. 39:945(1974)).

Superoxide dismuting activity. The SOD-like activities were measuredusing the xanthine/xanthine oxidase system as a source of O₂ ⁻ andferricytochrome c as its indicating scavenger (McCord et al, J. Biol.Chem. 244:6049 (1969)). O₂— was produced at the rate of 1.2 μM perminute and reduction of ferricytochrome c was followed at 550 nm. Assayswere conducted in presence of 0.1 mM EDTA in 0.05 M phosphate buffer (pH7.8). Rate constants for the reaction of the compounds were based uponcompetition with 0.10 μM cytochrome c, k_(cyt c)=2.6×10⁵ M⁻¹s⁻¹ (Butleret al, J. Biol. Chem. 257:10747 (1982)). All measurements were done at25° C. Cytochrome c concentration was at least 10³-fold higher than theconcentrations of the SOD mimics and the rates were linear for at leasttwo minutes, during which the compounds intercepted ˜100 equivalents ofO₂ ⁻, thus confirming the catalytic nature of O₂ ⁻ dismutation inpresence of the mimics.

Results

Despite increasing knowledge on the purification of water solubleporphyrins, the separation of halogenated uncharged porphyrins followedby f-alkylation and metallation still appeared easier for the successfulpreparation of MnCl_(x)TE-2-PyP⁵⁺ (Scheme A) (Richards et al, Inorg.Chem. 35:1940 (1996); Kaufman et al, Inorg. Chem. 34:5073 (1995)):

Synthesis of H₂T-2-PyP β-chlorinated derivatives. β-Chlorination ofH₂T-2-PyP was performed as described in the literature for H₂TPPanalogues, using N-chlorosuccinimide (NCS) in chloroform under refluxingconditions (Ochsenbein et al, Angew. Chem. Int. Ed. Engl. 33:348(1994)). The number of NCS equivalents used can be 4 or 6, depending onthe degree of substitution desired. (Table 3). The reaction can befollowed by TLC (silica gel) using a mixture ethanol/dichloromethane(5:95) as eluant (Table 3 and Scheme B). TABLE 3 H₂Cl_(x)T-2-PyP (x = 1to 4): R_(t), Soret band data and yields with 4 and 6 equivalents ofNCS. Yield (%)^(c) Porphyrin R_(t) ^(a) λnm (ε/10⁵ M⁻¹ cm⁻¹)^(b) 4 eq 6eq H₂T-2-PvP 0.43 418.4 β-Cl₁ 0.47 419.6 (2.74) 30 — β-Cl_(2a) 0.50421.4 (2.39) 10 5 β-Cl_(2b-2c) 0.53 421.4 (2.62) 20 10 β-Cl₃ 0.55 422.8(2.33) 10 15 β-Cl₄ 0.58 423.6 (2.13) 7 12^(a)TLC on silica with EtOH/CH₂Cl₂ (5:95) as eluant.^(b)in CHCl₃ (estimated errors for ε are within ⁼10%).^(c)in refluxing CHCl₃ during 6 hours (c˜2 μM).

Each compound was purified by chromatography on silica gel (WakogelC-300) using the same eluant. The structures of the main isomers wereidentified by mass spectrometry, and UV/VIS and ¹H-NMR spectroscopies(Table 3 and Scheme B). The bathochromic shift of the Soret band perchlorine on H₂T-2-PyP was only 1.3 nm compared to 3.5 nm reportedpreviously for H₂TPP derivatives (Table 3) (Hoffmann et al, Bull. Soc.Chem. Fr. 129:35 (1992); Chorghade et al, Synthesis 1320 (1996);Wijesekera et al, Bull. Chem. Fr. 133:765 (1996)). Only one of the threedichlorinated regioisomers (β-Cl_(2a) derivative) was purified bychromatography on silica gel. Its two other regioisomers (β-Cl_(2b) andβ-Cl_(2c) derivatives) exhibited the same R_(f). Preliminary resultsshowed that purification of H₂Br_(x)T-4-PyP (x=1 to 4) is moredifficult. Indeed, using the same TLC system, β-Br₁ and β-Br_(2a)derivatives both have the same R_(f), and no difference of R_(f) betweenβ-Br_(2b), β-Br_(2c), β-Br₃ and β-Br₄ derivatives was observed, showingclearly that, in this case, R_(f) depends on the number of pyrrolessubstituted and not on the number of protons substituted.

¹H-NMR identification of H₂T-2-PyP β-chlorinated derivatives. ¹H-NMRallowed the identification of the products of the substitution reaction(Table 4 and FIG. 5). As described in the literature for H₂TPPanalogues, the main regioisomer of H₂Cl₄T-2-PyP has chlorines inpositions 7, 8, 17, 18. Indeed, its ¹H-NMR spectrum shows an apparentsinglet (doublet with J lower than 2 Hz), corresponding to fourchemically equivalent β-protons coupled with the two pyrrolic protonswhich have lost their delocalization (Crossley et al, J. Chem. Soc.,Chem. Commun. 1564 (1991). Nevertheless, another less polar fraction(R_(f)=0.60) was identified, according to its mass spectrum, as amixture of other tetrachloro-regioisomers (¹H-NMR spectrumuninterpretable), representing approximately 50% by weight of both β-Cl₄fractions, and showing that the β-substitution is only partiallyregioselective. According to the ¹H-NMR spectrum of the correspondingH₂Cl₃T-2-PyP⁵⁺ fraction, there are no apparent other regioisomers. Thespectrum presents one singlet corresponding to the β-proton of themonosubstituted pyrrole and four doublets corresponding to the fourβ-protons of the two non-substituted pyrroles. Moreover, the asymmetryof this compound leads to a differentiation of the two NH protons.According to yields and ¹H-NMR spectra of H₂Cl_(2a)T-2-PyP (FIG. 5) andH₂Cl_(2b+2c)T-2-PyP, no predominant β-Cl₂ regioisomer was observed.Finally, the H₂Cl₁T-2-PyP spectrum shows one singlet and six doublets,but only one NH signal, suggesting that in this case the asymmetry istoo weak for the differentiation of the two NH protons. TABLE 4H₂Cl_(x)T-2-PyP(x = 1 to 4): ¹H-NMR data (porphyrin ring) in CDCl₃δ_(ppm) (mult.. Hz)^(a) H₂Cl₁T-2-PyP NH −2.91 (2H) CH 7.68 (s, 1H) 8.74(d, 1H, 5.5) 8.76 (d, 1H, 5.5) 8.76 (d, 1H, 6.0) 8.38 (d. 1H, 6.0) 8.90(d, 1H, 6.0) 8.94 (d, 1H, 6.0) H₂Cl_(2a)T-2-PyP NH −2.98 (2H) CH 8.70(s, 2H) 8.82 (d, 2H, 6.0) 8.91 (d, 2H, 6.0) H₂Cl_(2b)T-2-PyP^(b) NH−3.04 (2H) CH 8.59 (s, 2H) 8.73 (d, 2H, 6.0) 8.37 (d, 2H, 6.0)H₂Cl_(2c)T-2-PyP^(b) NH −2.84 (1H) −2.87 (1H) CH 8.61 (s, 2H) 8.73 (d,2H, <2.0) 8.93 (d, 2H, <2.0) H₂Cl₃T-2-PyP NH −3.08 (1H) −3.15 (1H) CH8.56 (s, 1H) 8.72 (d, 1H, 6.5) 8.76 (d, 1H, 6.5) 8.82 (d, 1H, 6.5) 8.88(d, 1H, 6.5) H₂Cl₄T-2-PyP NH −3.14 (2H) CH 8.74 (d, 4H, <2.0)^(a)chemical shifts in ppm expressed relative to TMS by setting CDCl₃ =7.24 ppm.^(b)one spectrum for the mixture of the two regioisomers (˜1:1 ratio).

N-ethylation and metallation. The N-ethylation of H₂T-2-PyP wasefficiently accomplished using ethyl-p-toluenesulfonate, diethylsulfateor iodoethane as reagents, but the high toxicity of diethylsulfate andthe low reactivity of iodoethane makes ethyl-p-toluenesulfonate (ETS)the best choice (Chen et al, J. Electroanal. Chem. 280:189 (1990);Kalyamasundaram, Inorg. Chem. 23:2453 (1984); Hambright et al, Inorg.Chem. 15:1314 (1976); Alder et al, Chem. Brit. 14:324 (1978);Perree-Fauvet et al, 52:13569 (1996)). Some authors prefer performingN-alkylation after metallation in order to protect the pyrrole nitrogens(Perree-Fauvet et al, Tetrahedron 52:13569 (1996)). However, with directtreatment on the present free ligands, no N-ethylation of the pyrrolenitrogens was observed (subsequent metallation in aqueous solution wascomplete). The completion of ethylation as well as metallation can befollowed by TLC (normal silica) using a highly polar eluant, a mixtureof an aqueous solution of saturated potassium nitrate with acetonitrile(Batinic-Haberle et al, J. Biol. Chem. 273:24521 (1998)). The yields ofthis step (NV-ethylation and metallation) were almost 100%(approximately 5% loss during the purification process). SinceN-ethylation (or N-methylation) limits the free rotation of thepyridinium rings, each compound is in fact a mixture of fouratropoisomers, and a further purification of each atropoisomer can beconsidered (Kaufmann et al, Inorg. Chem. 34:5073 (1995)). All themanganese porphyrins prepared had metal in the 3+ state as demonstratedby the 20 nm hypsochromic shift of the Soret band (accompanied by theloss of splitting) upon the reduction of the metal-center by ascorbicacid.

Electrochemistry. The metal-centered redox behavior of allmetalloporphyrin products was reversible. The half-wave potentials(E^(O) _(1/2)) were calculated as the average of the cathodic and anodicpeaks and are given in mV vs NHE (Table 5). The average shift perchlorine is +55 mV (Table 5), which is in agreement with the valuespreviously reported for H₂TPP derivatives (between −50 and +70 mV) (Senet al, Chem. Soc. Faraday Trans. 93:4281 (1997); Autret et al. J. Chem.Soc. Dalton Trans. 2793 (1996); Hariprasad et al, J. Chem. Soc. DaltonTrans. 3429 (1996); Tagliatesta et al, Inorg. Chem. 35:5570 (1996);Ghosh, J. Am. Chem. Soc. 117:4691 (1995); Takeuchi et al, J. Am. Chem.Soc. 116:9730 (1994); Binstead et al, Inorg. Chem. 30:1259 (1991);Giraudeau et al, J. Am. Chem. Soc. 101:3857 (1979)). This shift appearsto be higher (˜+65 mV) between 0 and 1, and between 2 and 3 chlorines(Table 5). E^(O) _(1/2) values of β-Cl_(2a) and the mixture β-Cl_(2b+2c)were not significantly different. The manganese redox state ofMnCl₄TE-2-PyP⁵ (E^(O) _(1/2)=+448 mV) and MnOBTMPyP⁴⁺ (E^(O) _(1.2)=+480mV) is 3+ and 2+, respectively. This difference may be explained bytheir difference in terms of redox potential (˜30 mV) but also bystructural considerations, for instance an increased distortion of theporphyrin ring in the case of MnOBTMPyP⁴⁺. (Batinic-Haberle et al, Arch.Biochem. Biophys. 343:225 (1997); Ochsenbein et al, Angew. Chem. Int.Ed. Engl. 33:348 (1994)). TABLE 5 MnCl_(x)TE-2-PyP⁵⁻(x = 1 to 4): Soretband data, redox potentials and SOD activities. Mn-porphyrin λnm (ε/10⁴M⁻¹ cm⁻¹)^(a) E°_(1/2) (Δ)^(b) IC₅₀/10⁻⁹ M^(c) k_(cat)/10⁷ M⁻¹ s⁻¹MnTE-2-PyP⁵⁻ 453.8 (14.0) +228 (71) 45 5.7 β-Cl₁ 455.6 (12.5) +293 (65)25 10 β-Cl_(2a) 456.4 (10.6) +342 (70) 20 13 β-Cl_(2b-2c) 457.4 (11.2)+344 (65) 20 13 β-Cl₃ 458.0 (9.5) +408 (67) 10 26 β-Cl₄ 459.2 (8.0) +448(79) 6.5 40 MnTM-4-PyP⁵⁻ +060 0.4 MnTM-2-PyP³⁻ +220 6.0 MnOBTMPyP⁴⁻ +48022 Cu•ZnSOD +260 200^(a)in H₂O (estimated errors for ε are within ⁼10%).^(b)mV vs NHE, with estimated errors of ⁼5 mV (Δ = peak to peakseparation), and in the following conditions: 0.5 mM porphyrin, 0.1 MNaCl, 0.05 M phosphate buffer (pH 7.8).^(c)concentration that causes 50% inhibition of cytochrome c reductionby O₂ ⁻(estimated errors are within ⁼10%).

Superoxide dismuting activities. SOD-like activities were measured asdescribed previously, based on competition with cytochrome c (McCord etal, J. Biol. Chem. 244:6049 (1969)). MnCl_(x)TE-2-PyP⁵⁺ SOD-likeactivities are reported in Table 5, IC₅₀ (M) representing theconcentration for one unit of activity (or the concentration that causes50% inhibition of cytochrome c reduction by O₂ ⁻) and k_(cat) (M⁻¹s⁻¹)representing the rate constant for the superoxide dismutation reaction.The SOD-like activity per mole of MnCl₄TE-2-PyP⁵ is approximately 2-, 7-and 100-fold higher than MnOBTMPyP⁴⁺, MnTM-2-PyP⁵⁺ and MnTM-4PyP⁵⁺,respectively (Faulkner et al, J. Biol. Chem. 269:23471 (1994);Batinic-Haberle et al, Arch. Biochem. Biophys. 343:225 (1997);Batinic-Haberle et al, J. Biol. Chem. 273:24521 (1998)). The SOD-likeactivity of MnCl₄TE-2-PyP⁵⁺ represents 20% of the activity of theCu,Zn-SOD enzyme on a molar basis (40% per active site considering thatthe enzyme has two active sites) (Klug-Roth et al, J. Am. Chem. Soc.95:2786 (1973)).

Test of stability. Each additional degree of chlorination increases theredox potential which is expected to be followed by the decrease in thepKa values of pyrrole nitrogens, as found for the series of meso-phenyland meso-pyridyl substituted porphyrins as well as for β-substitutedones (Worthington et al, Inorg. Nucl. Chem. Lett. 16:441 (1980); Kadishet al, Inorg. Chem. 15:980 (1976)). The pKa, as a measure of theligand-proton stability, is in turn a measure of the metal-ligandstability as well. Thus, the tetrachloro-compound is expected to be ofdecreased stability as compared to lesser chlorinated analogues. Thestability of MnCl₄TE-2-PyP⁵⁺ was tested by measuring its SOD-likeactivity in the presence of excess EDTA. In the presence of a 10²-foldexcess of EDTA, MnCl₄TE-2-PyP⁵⁺ (c=5×10⁻⁶M) maintains its activity forsixteen hours (at 25° C.). A loss of activity (˜25%) was observed afterforty hours, thus indicating the formation of some manganese—EDTAcomplex (K=10^(14.05)). These results confirm a relatively goodstability of MnCl₄TE-2-PyP⁵⁺ when compared to MnOBTMPyP⁴⁺ (T=10^(8.08))(Batinic-Haberle et al, Arch Biochem. Biophys. 343:225 (1997)).

Relationship between redox properties and SOD-like activities. TheCu,Zn-SOD enzyme is a dimer of two identical subunits, and thus has twoactive sites, which exhibit a redox potential close to the midpoint ofthe two half reaction values, as well as the same rate constants foreach half reaction (Scheme C and Table 5) (Ellerby et al, J. am. Chem.Soc. 118:6556 (1996); Klug-Roth, J. Am. Chem. Soc. 95:2786 (1973)):

On the other hand, previous studies of O₂ dismutation catalyzed byMnTM-4-PyP⁵⁺ (E^(O) _(1/2)=+60 mV), using pulse radiolysis and stoppedflow techniques, showed that the rate of the reduction of the metal byO₂— is 10²-fold to 10³-fold lower than the rate of reoxidation of themetal (Faraggi, Oxygen Radicals in Chemistry and Biology, Bors et al(Eds): Walter de Gruyter and Co.; Berlin, Germany 1984, p. 419; Lee etal, J. Am. Chem. Soc. 120:6053 (1998)). Whereas a peak of SOD-likeactivity somewhere between +200 and +450 mV was first expected, plottingk_(cat) vs E^(O) _(1/2) for MnCl_(x)TE-2-PyP⁵⁺ shows an exponentialincrease of the SOD-like activity, strongly suggesting that the limitingfactor is still the reduction of the metal. This hypothesis however mustbe confirmed by measuring the rates of each half reaction as catalyzedby each. MnCl_(x)TE-2-PyP⁵ compound. The relationship between activationfree energy (ΔG^(#)) for superoxide dismutation and free energy change(ΔG^(o)) for MnCl_(x)TE-2-PyP⁵⁺ redox is linear (slope ˜+0.2), clearlyshowing the predominance of kinetic over thermodynamic factors in thetheoretical optimal redox potential region (FIG. 6). According to thisbehavior, the activity of the Cu,Zn-SOD enzyme (k_(cat)=10⁹ M⁻¹s⁻¹ peractive site) may be reached at approximately E^(o) _(1/2)=+570 mV (FIG.3). However, due: to both steric (distortion of the porphyrin ring) andthermodynamic factors, introducing a higher degree of β-chlorination isexpected to stabilize the manganese in the 2+ redox state, and thus, asin the case of MnOBTMPyP⁴⁺, limiting the rate of the reoxidation of themetal as well as inducing Mn(II) dissociation (Batinic-Haberle et al,Arch. Biochem. Biophys. 343:225 (1997); Ochsenbein et al, Angew. Chem.Int. Ed. Engl. 33:348 (1994)).

Example VII

The ortho, meta and para isomers of manganese(III)5,10,15,20-tetrakis(N-methylpyridyl)porphyrin, MnTM-2-PyP⁵⁺,MnTM-3-PyP⁵⁺, and MnTM-4-PyP⁵⁺, respectively, were analyzed in terms oftheir superoxide dismutase (SOD) activity in vitro and in vivo. Theimpact of their interaction with DNA and RNA on the SOD activity in vivoand in vitro was also analyzed. Differences in their behavior are due tothe combined steric and electrostatic factors. In vitro catalyticactivities are closely related to their redox potentials. The half-wavepotentials (E_(1/2)) are +0.220 mV, +0.052 mV and +0.060 V vs normalhydrogen electrode (NHE), while the rates of dismutation (k_(cat)), are6.0×10⁷ M⁻¹s⁻¹, 4.1×10⁶ M⁻¹s⁻¹ and 3.8×10⁶ M⁻¹s⁻¹ for the ortho, metaand para isomers, respectively.

However, the in vitro activity is not a sufficient predictor of in vivoefficacy. The ortho and meta isomers, although of significantlydifferent in vitro SOD activities, have fairly close in vivo SODefficacy due to their similarly weak interactions with DNA. In contrast,due to a higher degree of interaction with DNA, the para isomerinhibited growth of SOD-deficient Escherichia coli. For details, seeBatinic-Haberle et al, J. Biol. Chem. 273(38):24521-8 (Sep. 18, 1998).

Example VIII Metalloporphyrins are Potent Inhibitors of LipidPeroxidation

Materials and Methods

L-Ascorbic acid, n-butanol, butylated hydroxytoluene, cobalt chloride,iron(II) chloride, phosphoric acid (85%), sodium hydroxide, potassiumphosphate, tetrabutylammonium chloride, and 1,1,3,3-tetramethoxypropanewere purchased from Sigma (St. Louis, Mo.). Acetone, concentratedhydrochloric acid, 4,6-dihydroxy-2-mercaptopyrimidine (thiobarbituricacid), NH₄ PF₆, zinc chloride, 5,10,15,20-tetrakis(4-benzoic acid)porphyrin (H₂TBAP)*,5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrin (H₂TM-4-PyP), andTrolox were purchased from Aldrich (Milwaukee, Wis.). Ferric5,10,15,20-tetrakis(4-benzoic acid) porphyrin (FeTBAP) was purchasedfrom Porphyrin Products (Logan, Utah).5,10,15,20-tetrakis(N-methylpyridinium-2-yl)porphyrin (H₂TM-2-PyP) waspurchased from MidCentury Chemicals (Posen, Ill.). (+)-Rutin waspurchased from Calbiochem (La Jolla, Calif.). Manganese chloride waspurchased from Fisher (Fair Lawn, N.J.) and ethanol USP was purchasedfrom AAPER Alcohol and Chemical Co. (Shelbville, Ky.). All solutionswere prepared in Milli-Q Plus PF water (Millipore, Bedford, Mass.).Also known as 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin (H₂TCPP)

Preparation and Analysis of Metalloporphyrins

The metalloporphyrins MnTBAP, CoTBAP and ZnTBAP were made using methodsdescribed previously (Day et al, J. Pharmacol. Exp. Ther. 275:1227(1995)). MnTM-4-PyP, CoTM-4-PyP and ZnTM-4-PyP were synthesized by thefollowing method. A 1.5 molar excess of manganese, cobalt or zincchloride was mixed with H₂TM-4-PyP that was dissolved in de-ionizedwater. The reaction mixture was heated to 80° C. and metal ligation wasfollowed spectrophotometrically (UV-2401PC, Shimadzu, Columbia, Md.).Excess metal was removed by passing the mixture through a columncontaining Bio-Gel P-2 (BioRad, Richmond, Calif.) that selectivelyretained MnTM-4-PyP. MnTM-4-PyP was eluted with 0.01 N HCl afterextensive washing of the column with water. MnTM-4-PyP, CoTM-4-PyP andZnTM-4-PyP were characterized in terms of their reported Soret bands.The Soret band for MnTM-4-PyP is at 463 nm with an extinctioncoefficient of (ε) 1.3×10⁵ M⁻¹cm⁻¹, the Soret band for ZnTM-4-PyP is at437 nm with an extinction coefficient of (ε)=2.0×10⁵ M⁻¹cm⁻¹ (Pasternacket al, Inorg. Chem. 12:2606 (1973)) and the Soret band for CoTM-4-PyP isat 434 nm with an extinction coefficient of (ε)=2.15×10⁵ M⁻¹cm⁻¹(Pasternack et al, Biochemistry 22:2406 (1983)). Manganeseβ-octabromo-meso-tetrakis-(N-methylpyridinium-4-yl)porphyrin.(MnOgTM-4PyP) was synthesized as previously described (Batinic-Haberleet ah, Arch. Biochem. Biophys. 343:225 (1997)) and has a Soret band at490 nm with an extinction coefficient (ε)=8.56×10⁴ M⁻¹ cm⁻¹. H₂TM-2-PyPwas metallated with a 1:20 porphyrin to manganese ratio in water (pH>11)at room temperature. Upon completion of metallation, MnTM-2-PyP wasprecipitated by the addition of a concentrated aqueous solution of NH₄PF₆. The precipitate was washed with 2-propanol:diethyl ether (1:1) anddried in vacuo at room temperature. The PF₆— salt of MnTM-2-PyP wasdissolved in acetone, filtered and a concentrated acetone solution oftetrabutylammonium chloride was added until the porphyrin hadprecipitated as its chloride salt. The precipitate was washed withacetone and dried in vacuo at room temperature. The Soret band forMnTM-2-PyP was found at 453 nm with an extinction coefficient(O)=1.29×10⁵ M⁻¹cm⁻¹.

Preparation of Rat Brain Homogenates

Frozen adult Sprague-Dawley rat brains (Pel-Freez, Rogers, Ark.) werehomogenized with a polytron (Turrax T25, Germany) in 5 volumes of icecold 50 mM potassium phosphate at pH 7.4. Homogenate proteinconcentration was determined with the Coomassie Plus protein assay(Pierce, Rockford, Ill.) using bovine serum albumin as a standard. Thehomogenate volume was adjusted with buffer to give a final proteinconcentration of 10 mg/ml and frozen as aliquots at 80° C.

Oxidation of Rat Brain Homogenates

Rat brain homogenates (2 mg protein) were incubated with varyingconcentrations of antioxidant at 37° C. for 15 minutes. Oxidation of therat brain homogenate was initiated by the addition of 0.1 ml of afreshly prepared anaerobic stock solution containing iron(II) chloride(0.25 mM) and ascorbate (1 mM) as previously reported (Braughler et al,J. Biol. Chem. 262:10438 (1987)). Samples (final volume 1 ml) wereplaced in a shaking water bath at 37° C. for 30 minutes. The reactionswere stopped by the addition of 0.1 ml of a stock butylatedhydroxytoluene (60 mM) solution in ethanol.

Lipid Peroxidation Measurement

The concentration of thiobarbituric acid reactive species (TBARS) in ratbrain homogenates was used as a index of lipid peroxidation (Bernhem etal, J. Biol. Chem. 174:257 (1948); Witz et al, J. Free Rad. Biol. Med.2:33 (1986); Kikugawa et al, Anal. Biochem. 202:249 (1992); Jentzsch etal, Free Rad. Biol. Med. 20P251 (1996)). Malondialdehyde standards wereobtained by adding 8.2 μl of 1,1,3,3-tetramethoxypropane in 10 ml of0.01 M HCl and mixing for 10 minutes at room temperature. This stock wasfurther diluted in water to give standards that ranged from 0.25 to 25μM. Samples or standards (200 μl) were acidified with 200 μl of 0.2 Mphosphoric acid in 1.5 ml locking microfuge tubes. The color reactionwas initiated by the addition of 25 μl of a 0.11M thiobarbituric acidsolution and samples were placed in a 90° C. heating block for 45minutes. TBARS were extracted with 0.5 ml of n-butanol by vortexingsamples for 3 minute and chilling on ice for 1 minute. The samples werethen centrifuged at 12,000×g for 3 minutes, 150 μl aliquots of then-butanol phase were placed in each well of a 96-well plate and read at535 nm in a Thermomax platereader (Molecular Devices, Sunnyvale, Calif.)at 25° C. Sample absorbencies were converted to MDA equivalencies (μM)by extrapolation from the MDA standard curve. None of the antioxidantsat concentrations employed in these studies affected the reaction of MDAstandards with thiobarbituric acid and reactions without TBA were usedas subtraction blanks.

Statistical Analyses

Data were presented as their means ±SE. The inhibitory concentration ofantioxidants that decreased the degree of lipid peroxidation by 50%(IC₅₀) and respective 95% confidence intervals (CI) were determined byfitting a sigmoidal curve with variable slope to the data (GraphPadPrizm, San Diego, Calif.).

Results

Comparison of Metalloporphyrins with Other Antioxidants inIron/Ascorbate-Mediated Lipid peroxidation.

The objective of these studies was to investigate whethermetalloporphyrins could inhibit lipid peroxidation and to compare theirto potencies with those of previously characterized antioxidants thatinclude enzymatic antioxidants (SOD and catalase) and non-enzymaticantioxidants (water soluble vitamin E analog, trolox, and plantpolyphenolic flavonoid, rutin) (FIG. 7). The time course of lipidperoxidation was determined in rat brain homogenates using iron andascorbate as initiators of lipid oxidation and the formation ofthiobarbituric reactive species (TBARS) as an index of lipidperoxidation. A linear increase in the formation of TBARS occurredbetween 15 to 90 minutes of incubation at 37° C. (FIG. 8). Based on thisresult, an incubation time of 30 minutes was selected to test theability of metalloporphyrins and other antioxidants to inhibit lipidperoxidation. (FIG. 9). Of the agents tested, the manganese porphyrinsthat have the highest SOD activities, MnOBTM-4-PyP and MnTM-2-PyP, werefound to be the most potent lipid peroxidation inhibitors withcalculated IC₅₀s of 1.3 and 1.0 μM respectively. (Table 6). BovineCuZnSOD was moderately active with a calculated IC₅₀ of 15 μM whiletrolox and rutin were much less potent with calculated IC₅₀s of 204 and112 μM, respectively. In this system, catalase (up to concentrations of1 mg/ml) did not inhibit iron/ascorbate-initiated lipid peroxidation.TABLE 6 Comparison of Antioxidant Properties Lipid Peroxidation^(c) SODRedox Potential IC₅₀ 95% Cl Antioxidants (U/mg)^(a) (E_(1/2), V)^(b)[μM] [μM] CuZnSOD 5,100   +0.35 15 13-17 Trolox — — 204 135-308 Rutin —— 113  99-129 MnTM-2-PyP 8,500   +0.22 1.0 0.4-2.2 MnOBTM-4-PyP 18,460  +0.48 1.3 0.8-2.2 MnTM-4-PyP 547 +0.06 16 12-22 MnTBAP 179 −0.19 2923-37 CoTM-4-PyP 113 +0.42 17 14-22 CoTBAP  24 +0.20 21 13-33 FeTBAP  24+0.01 212 144-311 ZnTM-4-PyP trace — 241 159-364 ZnTM-2-PyP trace — 591423-827 ZnTBAP trace — 843  428-1660^(a)Unit of SOD activity defined as the amount of compound that inhibitsone half the reduction of cylochrome c or photoreductlon of NBT.^(b)Metal centered redox potentials vs NHE (Mn⁺³/Mn⁺²; Co⁺³/Co⁺²;Fe⁺³/Fe⁺²). If not otherwise specified, E_(1/2) were obtained at pH 7.8.^(c)The amount of thiobarbaturic acid reactive substances produced in arat brain homogenate by 30 minutes of incubation of iron and ascorbate.Effect of Different Metal Chelates on the Ability of Porphyrins toInhibit Lipid Peroxidation.

A wide range of metals can be covalently ligated by porphyrins and thatconfers different redox potentials and SOD activities (Table 6). Theability of different metal chelates to influence a porphyrin's abilityto inhibit lipid peroxidation was tested. Several different metalanalogs of TBAP were examined in the iron/ascorbate-initiated lipidperoxidation model (FIG. 10). Both the manganese and cobalt TBAP analogshad similar efficacy with calculated IC₅₀ of 29 and 21 μM, respectively.The FeTBAP analog was an order of magnitude less potent with acalculated IC₅₀ of 212 μM. The ZnTBAP analog was much less active thanthe other metal analogs with a calculated IC₅₀ of 946 μM. This potencydifference between the zinc and the other metals reflects the importanceof metal centered verses ring structure redox chemistry since zinc cannot readily change its valence. The ranked potencies of testedmetalloporphyrins based on IC₅₀s were as follows:MnTM-2-PyP=MnOBTM-4-PyP>MnTM-4-PyP=CoTM-4-PyP>CoTBAP=MnTBAP>FeTBAP=ZnTM-4-PyP>ZnTM-2-PyP>ZnTBAP.

Comparison of a Series of Tetrakis N-Methylpyridyl Porphyrin (TMPyP)Analogs as Inhibitors of Lipid Peroxidation.

Recently, several manganese analogs of N-methylpyridyl porphyrins havebeen found to possess large differences in SOD activities (Table 6).MnTM-2-PyP and MnTM-4-PyP differ structurally with respect to theposition of the N-methylpyridyl group to the porphyrin ring (ortho vspara) as well as in SOD activity by a factor of six. Substitution ofzinc in these porphyrin analogs results in loss of SOD activity. TheseTMPyP analogs to were compared for their ability to inhibit lipidperoxidation (FIG. 11). The movement of the N-methylpyridyl group fromthe para- to the ortho-position in the manganese porphyrin resulted in a15-fold increase in potency. Since MnTM-2-PyP possesses a more positiveredox potential than MnTM-4-PyP (+0.22 vs +0.06, respectively), thisdata suggests that both the redox potential and the related SOD activitymay contribute to the increased potency of the MnTM-2-PyP analog.

Example IX Demonstration that Mn TE-2-PyP can be Effectively Used toAttenuate Oxidant Stress Mediated Tissue Injury

The ability of Mn TE-2-PyP to attenuate injury associated with 60minutes of global ischemia followed by 90 minutes of reperfusion wasassessed in an isolated, perfused mouse liver model. Excised livers wereperfused with a buffered salt solution for 15 minutes after which themetalloporphyrin was introduced into the perfusate and the liverperfused in a recirculating system for an additional 15 minutes. Thelivers were then rendered globally ischemic under normal thermicconditions for 60 minutes. Following the ischemic period the livers wereperfused for 90 minutes with perfusate supplemented with 10 μm MnTE-2-PyP. In this model the ischemia/reperfused livers have a markedrelease of hepatocellular enzymes, aspartate transaminase, alaninetransaminase, and lactate dehydrogenase during the first 2½ minutes ofreperfusion. This is followed by a progressive release of hepatocellularenzymes indicating hepatocellular injury over the 90 minute perfusionperiod. Administration of Mn TE-2-PyP was highly efficacious inattenuating the liver injury, blocking virtually all of the acutehepatocellular enzyme release and blocking progressive hepatocellularenzyme release over the 90 minute perfusion period. At the end of theexperiments liver is treated with the metalloporphyrin. It hasdemonstrated excellent oxygen consumption and a normal perfusionpattern. They remain firm and with a normal texture to gross morphologicexamination. Livers with no drug treatment did not consume oxygennormally and became edematous, soft, and had a mottled appearanceconsistent with poor perfusion.

Example X Effects of Mn TM-2-PyP on Vascular Tone

Rats were anesthetized and a femoral vein and carotid artery werecannulated. While blood pressure was monitored by the carotid artery, MnTM-2-PyP was injected i.v. at doses ranging from 0.1 to 3.0 mg/kg. Meanarterial pressure fell from 100-125 mmHg to 50-60 mmHg within five toten minutes. The effect was transient, lasting up to 30 minutes at dosesof 0.1 to 0.25 mg/kg. At doses of 1-3 mg/kg the effect was prolonged,lasting up to two hours. The effect can be blocked by administration ofinhibitors of nitric oxide synthase demonstrating that the role of MnTM-2-PyP is being modulated by nitric oxide. Scavenging of superoxide invascular walls would potentiate the effects of nitric oxide producinghypotension.

Example XI Regulation of Airway Reactivity Using Mn TM-2-PyP

Mice were sensitized by intraperitoneal injection of ovalbumin twice, 14days apart. Fourteen days after the second i.p. injection they werechallenged with aerosolized ovalbumin daily for three days. Forty-eighthours after the third inhalation of ovalbumin they were given a 1 minutemethacholine challenge and airway hyperreactivity followed using a Buxcobody plethysmograph. Significant increases in airway resistance asmeasured by the PENH index occurred at doses of 20, 30 and 40 mg/ml ofmethacholine. At all doses of methacholine prior intratrachealinstillations of 2 μg Mn TM-2-PyP given daily for 4 days resulted in astatistically significant reduction in the airway hyperreactivity. Thisdose of in TM-2-PyP is equivalent to 0.8 mg/kg whole body dose.

Example XII Treatment of Bronchopulmonary Dysplasia Using Mn TE-2-PyP

Neonatal baboons were delivered prematurely by Caesarian section andthen treated either with 100% oxygen or only sufficient PRN FIO₂ tomaintain adequate arterial oxygenation. To establish the model, thirteen100% oxygen treated animals and twelve PRN control animals were studied.Treatment with 100% oxygen results in extensive lung injury manifestedby days 9 or 10 of exposure and characterized by delayedalveolarization, lung parenchymal inflammation, and poor oxygenation.This is characteristic of the human disease, bronchopulmonary dysplasiaand is thought to be mediated, at least in part, by oxidative stress onthe developing neonatal lung. In a first trial of Mn TE-2-PyP, aneonatal baboon was delivered at 140 days gestation and placed in 100%oxygen. The animal received 0.5 mg/kg/24 hr Mn TE-2-PyP qd given i.v. ina continuous infusion over the entire 10 day study period. This animalshowed marked improvement of the oxygenation index. There was noevidence of clinical decompensation of the lungs at days 9 and 10. Lungpathology demonstrated absence of inflammation and a marked decrease inthe lung injury found in the prior animals treated with 100% oxygenunder identical conditions. This suggests that Mn TE-2-PyP can be usedto treat oxidant stress in the premature newborn.

All documents cited above are hereby incorporated in their entirety byreference. Appln. No. 60/064,116, filed Nov. 3, 1997, is alsoincorporated in its entirety by reference.

One skilled in the art will appreciate from a reading of this disclosurethat various changes in form and detail can be made without departingfrom the true scope of the invention.

1. A compound of formula

or pharmaceutically acceptable salt thereof, wherein each R is,independently, a C₁-C₈ alkyl group, and each P is, independently, anelectron withdrawing group or hydrogen, wherein when each R is methyland each P is hydrogen, said compound is completed with a metal selectedfrom the group consisting of manganese, iron, copper, cobalt, nickel orzinc.
 2. The compound according to claim 1 where each R is independentlya C₁-C₄ alkyl group.
 3. The compound according to claim 2 wherein each Ris, independently, a methyl, ethyl or isopropyl group.
 4. The compoundaccording to claim 3 wherein each R is, independently, a methyl or anethyl group.
 5. The compound according to claim 1 wherein each P is,independently, hydrogen or an electron withdrawing group selected fromthe group consisting of —NO₂, a halogen, a nitrile, a vinyl group and aformyl group.
 6. The compound according to claim 1 wherein at least oneP is a halogen.
 7. The compound according to claim 1 wherein one or twoP's are formyl groups and the remaining P's are hydrogen.
 8. Thecompound according to claim 1 wherein one P is a formyl group and theremaining P's are hydrogen.
 9. The compound according to claim 1 whereinone or two P's are —NO₂ and the remaining P's are hydrogen.
 10. Thecompound according to claim 1 wherein said compound is completed with ametal selected from the group consisting of manganese, iron, copper,cobalt, nickel or zinc.
 11. The compound according to claim 10 whereinsaid compound is completed with manganese.
 12. The compound according toclaim 1 wherein each R is a methyl or ethyl group, each P is a hydrogen,and said compound is completed with manganese.
 13. The compoundaccording to claim 1 wherein each R is a methyl or ethyl group, at leastone 2 is Br and the remaining P's are hydrogen and said compound iscomplexed with manganese.
 14. The compound according to claim 1 whereinsaid compound is a mixture of atrocoisomers αααα, αααβ, ααββ and αβαβ.15. The compound according to claim 1 wherein said compound is a mixtureof αααβ and αααα atropoisomers.
 16. A method of protecting cells fromoxidant-induced toxicity comprising contacting said cells with aprotective amount of a compound of formula

or pharmaceutically acceptable salt thereof, wherein each R is,independently, a C₁-C₈ alkyl group, and each P is, independently, anelectron withdrawing group or hydrogen.
 17. The method according toclaim 16 wherein said compound is complexed with a metal selected fromthe group consisting of manganese, iron, copper, cobalt, nickel or zinc.18. The method according to claim 16 wherein said cells are mammaliancells.
 19. A method of treating a pathological condition of a patientresulting from oxidant-induced toxicity comprising administering to saidpatient an effective amount of a compound of formula

or pharmaceutically acceptable salt thereof, wherein each R is,independently, a C₁-C₈ alkyl group, and each P is, independently, anelectron withdrawing group or hydrogen.
 20. The method according toclaim 19 wherein said compound is completed with a metal selected fromthe group consisting of manganese, iron, copper, cobalt, nickel or zinc.21. A method of treating a pathological condition of a patient resultingfrom degradation of NO. or a biologically active form thereof,comprising administering to said patient an effective amount of acompound of formula

or pharmaceutically acceptable salt thereof, wherein each R is,independently, a C₁-C₈ alkyl group, and each P is, independently, anelectron withdrawing group or hydrogen.
 22. The method according toclaim 21 wherein said compound is complexed with a metal selected fromthe group consisting of manganese, iron, copper, cobalt, nickel or zinc.23. A method of treating a patient for inflammatory lung diseasecomprising administering to said patient an effective amount of acompound of formula

or pharmaceutically acceptable salt thereof, wherein each R is,independently, a C₁-C₈ alkyl group, and each P is, independently, anelectron withdrawing group or hydrogen.
 24. The method according toclaim 23 wherein said compound is complexed with a metal selected fromthe group consisting of manganese, iron, copper, cobalt, nickel or zinc.25. The method according to claim 24 wherein said metal is manganese.26. The method according to claim 23 wherein said inflammatory lungdisease is a hyper-reactive airway disease.
 27. The method according toclaim 23 wherein said inflammatory lung disease is asthma.